1. Field of the Invention
The present invention relates generally to making immunoassay of human granulocyte elastase and, more particularly, to a method for precisely assaying the amount of human granulocyte elastase in the specimen to be assayed as the total amount of human granulocyte elastase, when that specimen contains a mixture of free elastase with an elastase-inhibitor complex as well as how to use it clinically.
2. Statement of the Prior Art
Human granulocytes are located in the forefront of the biophylaxis mechanism, gathering on the site of infection at the very initial stage of invasion and releasing proteases and active enzymes for decomposition, degradation and sterilization of foreign matters and bacteria. Of the released proteases, elastase has the strongest action and so holds a central position of the biophylaxis mechanism. However, on the other hand, this enzyme is too low in substrate specificity to decompose various connective tissue components (elastin, collagen, proteoglycan, etc.), inducing destruction of the living body's tissue due to digestion and degradation of constructive proteins.
Usually, a living body has in its blood a large amount of inhibitors, like .alpha..sub.1 -antitrypsin, so as to protect the tissue not subject to invasion against the strong digestive actions of enzymes; that is, even when elastase released from granulocytes gathering on the site of invasion is diffused throughout the living body by blood circulation, elastase-inhibitor complexes are immediately formed, whereby the activity of elastase is deactivated to prevent the tissue from being destructed more than required.
The determination of human granulocyte elastase playing such a role is considered very effective for diagnosis of inflammation or to clear up the cause of inflammation, treat it and judge recuperation, and a method of making imminoassay of the quantity of granulocyte elastase in blood is set forth in JP-A-57-551. According to this method, a elastase-inhibitor complex is immunologically assayed, using the first antibody an antibody for human granulocyte elastase and as the second antibody an enzyme labeled antibody for an inhibitor.
However, this method is used only for measuring elastase-inhibitor complexes; in other words, it lends itself fit for where circulating blood which contains the released human granulocyte elastase only in the form of an elastase-inhibitor complex is used as the specimen to be assayed. That is, a serious problem with this method is that when immunologically assaying specimens comprising tissular mucus obtained from the site of inflammation, which contains small amounts of inhibitors and in which free elastase is mixed with elastase-inhibitor complexes, the quantity of human granulocyte elastase cannot precisely be detected.
This is also true of when immunoassay is made using as the first antibody an antibody for human granulocyte elastase and as the second antibody an enzyme labeled antibody an inhibitor. In other words, the precise quantity of human granulocyte elastase cannot be detected, because there is a difference in the reactivities of said antibodies with free elastase and elastase-inhibitor complexes.
In view of the state-of-the-art problems mentioned above, the inventors have intensively studied of how to assay the precise quantity of human granulocyte elastase, even when free elastase is mixed with elastase-inhibitor complexes, and have accomplished the present invention.